Many Pseudomonas strains produce antifungal metabolites that have been implicated in the control of fungal pathogens in the rhizosphere. For example, Howell et al. (Phytopathology 69: 480-482 (1979)) disclose a strain of Pseudomonas fluorescens that produces an antibiotic substance antagonistic to Rhizoctonia solani. In addition, other strains of Pseudomonas fluorescens having enhanced biocontrol activity against plant pathogenic fungi such as Rhizoctonia and Pythium have been more recently been disclosed. Pseudomonas fluorescens strain CGA267356 (also known as both MOCG134 and BL915) has been shown to be effective in controlling plant pathogenic fungi such as Rhizoctonia and Pythium. Strain CGA267356 is one of the subjects of U.S. Pat. No. 5,348,742, incorporated herein by reference. Two mutants of CGA267356, strains CGA321730 (a.k.a. MOCG134-8392) and CGA319115, have been constructed and shown to demonstrate even better biological control (biocontrol) of these phytopathogens. CGA321730 and CGA319115 are the subject of U.S. Pat. No. 5,496,547, incorporated herein by reference.
A particularly effective antibiotic against fungal pathogens is pyrrolnitrin, which is biosynthesized from tryptophan. Pyrrolnitrin is a phenylpyrrole derivative with strong antibiotic activity that has been shown to inhibit a broad range of fungi. Pyrrolnitrin was originally isolated from Pseudomonas pyrrocinia, but has since been isolated from Myxococcus species, Burkholdaria species, and several other Pseudomonas species such as P. fluorescens. The compound has been reported to inhibit fungal respiratory electron transport and uncouple oxidative phosphorylation. It has also been proposed that pyrrolnitrin causes generalized lipoprotein membrane damage. U.S. patent application Ser. No. 08/729,214, incorporated herein by reference, describes the cloning and characterization of the pyrrolnitrin biosynthesis genes from P. fluorescens, P. pyrrocinia, Burkholdaria cepacia, and Myxococcus fulvus.
Two genes have been isolated from strain CGA267356 that encode proteins that regulate the synthesis of several antifungal compounds produced by the strain, including pyrrolnitrin. These are the lemA gene and gacA (a.k.a. gafA) gene that encode sensor kinase and response regulator proteins, respectively, which function as a typical two-component bacterial regulatory system. In a normal regulatory system and under the proper conditions, the LemA protein phosphorylates GacA. In the phosphorylated state, GacA activates transcription of genes involved in the synthesis of antifungal compounds, i.e, the pyrrolnitrin biosynthesis genes. The lemA and gacA genes and their use to activate biocontrol activity in biocontrol strains are described in U.S. Pat. No. 5,670,350, incorporated herein by by reference. The lemA and gacA regulatory genes and the pyrrolnitrin biosynthetic genes have been utilized to genetically modify P. fluorescens strains to construct altered strains that demonstrate enhanced production of antifungal metabolites, i.e. pyrrolnitrin, and accordingly enhanced biocontrol activity.